Chapman Emma J, Williams Sarah V, Platt Fiona M, Hurst Carolyn D, Chambers Philip, Roberts Paul, Knowles Margaret A
Cancer Research UK Clinical Centre, St James's University Hospital, Leeds LS97TF, UK.
Genes Chromosomes Cancer. 2009 Aug;48(8):694-710. doi: 10.1002/gcc.20672.
Much progress has been made in identifying the molecular genetic alterations that occur in bladder cancer. However, in many cases the genes targeted by these alterations are not known. Telomerase immortalized human urothelial cells (TERT-NHUC) are a useful resource for in vitro studies of genes involved in urothelial transformation. When cultured under standard conditions they remain genetically stable but when cultured under low-density conditions they exhibit genetic instability and acquire chromosomal alterations. TERT-NHUC from three donors were cultured at low plating density and examined at four time-points during a culture period of 600 days. Analyses included population doubling kinetics, array-based CGH (aCGH), chromosome counts, fluorescence in situ hybridization (FISH), mutation analysis, Affymetrix gene expression analysis, Western blotting for p16, anchorage-independent growth and tumorigenicity assays. Alterations acquired during continued culture of TERT-NHUC at low density (TERT-NHUC-L) included some observed in urothelial carcinoma (UC) cell lines and primary UC. Examination of gene expression in TERT-NHUC with distinct acquired genetic aberrations may pinpoint genes targeted by these alterations. Data from an aCGH study of UC cell lines and primary tumors were examined for changes in chromosomal regions that also showed alterations in TERT-NHUC-L. Loss of a region on 2q including BOK was identified in UC cell lines and primary tumors. DNER and FRAS1 were identified as potential candidate genes, whose expression is altered independently of the acquisition of any genetic event.
在识别膀胱癌中发生的分子遗传改变方面已经取得了很大进展。然而，在许多情况下，这些改变所靶向的基因尚不清楚。端粒酶永生化人尿路上皮细胞（TERT-NHUC）是用于尿路上皮转化相关基因体外研究的有用资源。在标准条件下培养时，它们保持遗传稳定，但在低密度条件下培养时，它们表现出遗传不稳定并获得染色体改变。来自三名供体的TERT-NHUC在低接种密度下培养，并在600天的培养期内的四个时间点进行检查。分析包括群体倍增动力学、基于芯片的比较基因组杂交（aCGH）、染色体计数、荧光原位杂交（FISH）、突变分析、Affymetrix基因表达分析、p16的蛋白质印迹、非锚定依赖性生长和致瘤性测定。在低密度下持续培养TERT-NHUC（TERT-NHUC-L）期间获得的改变包括在尿路上皮癌细胞系和原发性尿路上皮癌中观察到的一些改变。检查具有明显获得性遗传畸变的TERT-NHUC中的基因表达可能会确定这些改变所靶向的基因。对尿路上皮癌细胞系和原发性肿瘤的aCGH研究数据进行检查，以寻找在TERT-NHUC-L中也显示改变的染色体区域的变化。在尿路上皮癌细胞系和原发性肿瘤中发现2号染色体上包括BOK的一个区域缺失。DNER和FRAS1被确定为潜在的候选基因，其表达独立于任何遗传事件的获得而改变。
